The present invention relates to an efficient new method of protoplast culture.
All organisms, be it plants or animals, reproduce sexually by the fusion of male and female gametes, each containing a single set of chromosomes from either parent and this is possible only in the compatible and related species. Wide crosses are not common without human intervention. However, plant protoplast culture provides a unique system, wherein each protoplast has the potential to give rise to a whole plant and two or more protoplasts can be induced to produce a hybrid (fusion between nuclei) or a cybrid (fusion between cytoplasm of one and the nucleus of another). This technique is used to overcome sexual incompatibility barriers through the production of unique somatic hybrids involving vegetative cells. The lack of cell wall barriers offers significant advantages for the introduction of genetically engineered foreign DNA into the naked cellsxe2x80x94that are protoplasts.
Hanstein for the first time used the term protoplast in 1880 (cf. Cocking E. C. 1972. Plant cell protoplasts, isolation and development. Ann. Rev. Plant Physiol. 23: 29-50). The isolation of protoplasts from plant cells was first achieved by Klercker by microsurgery on plasmolysed cells in 1892 (cf. Cocking E. C. 1972. Plant cell protoplasts, isolation and development. Ann. Rev. Plant Physiol. 23: 29-50).). With refinements in the technique, protoplasts were beginning to be isolated in large numbers by enzymatic removal of cell wall as pioneered by Cocking in 1960 (A method for isolation of plant protoplasts and vacuoles. Nature 187: 927). The plant species, condition under which plants are grown, plant age, method of protoplast isolation and protoplast culture are often critical for sustained division of protoplasts. Therefore, there are no standard methods for the isolation and culture of protoplasts. Considerable success has been achieved over the past two decades, when a number of techniques have been employed for the culture of protoplasts of numerous crop species.
In one such method (Binding H. 1974. Cell cluster formation by leaf protoplasts from axenic cultures of haploid Petunia hybrida L. Plant Sci. Lett. 2(3): 185-187), the protoplasts at a desired density are suspended in the optimum quantity of liquid medium in a petri dish and incubated at 25-28xc2x0 C., generally in the dark or diffused light. Such a method provides opportunity to gradually change the osmolarity of the medium for better protoplast growth. But in this method, protoplasts generally tend to aggregate and some even degenerate which adversely affects the dividing protoplasts. This method also suffers from the fact that it requires relatively larger volume of protoplast suspension. Inadequate aeration also adversely affect protoplast growth.
Another method is the hanging/sitting drop culture method (Kao K. N., Keller W. A. and Miller R. A. 1970. Cell division in newly formed cells from protoplasts of soybean. Exp. Cell Res. 62: 338). In this method, small drops (40-100 l) of protoplast suspension are placed on the inner side of the lid of a petri dish so that the drops containing the protoplasts are hanging from the lid. The drops can also be placed at the bottom of the petri dish. Fresh medium can be added in small volumes whenever required. However, this method is generally employed where protoplast yield is low and also requires dexterous handling as a slight shaking disturbs the hanging drop.
Yet another technique used for protoplast culture is the micro-chamber culture method (Bawa S. B. and Torrey J. G. 1971. xe2x80x9cBuddingxe2x80x9d and nuclear division in cultured protoplast of corn, Convolvulus and onion. Bot. Gaz. 132: 420) which is similar to drop culture method except that in this case a cavity microslide or a micro-chamber is used in order to follow the development of individual protoplasts. Here, either a conditioned medium is used or else a feeder layer is required for protoplast growth. However, this method suffers from the defect of quick drying and requires special efforts to ensure aeration.
In yet another method called Micro-drop Array (M.D.A.) technique (Potrykus I., Harms C. T. and Lorz H. 1979. Multiple-drop array (MDA) technique for the large-scale testing of culture media variations in hanging microdrop cultures of single cell systems. 1. The technique. Plant Sci. Lett. 14:231), which is usually employed to test the response of protoplasts to different culture media under varying combinations and permutations. The drop size is reduced to 40 l to accommodate many drops per petri dish. This method provides opportunity to use smaller number of petri dishes. However in this method it is not easy to handle small drops where the size of the drop is too small and it tends to dry quickly.
Another method used is the Micro-droplet Culture Technique (Gleba Y. Y. 1978. Microdroplet cultures: tobacco plants from single mesophyll protoplasts. Naturwissenschaften 65: 158), wherein the size of culture droplet is reduced to about 0.25 to 0.50 l so that each droplet containing only one protoplast is placed separately in numbered wells of special Cuprak petri dishes. However, since only one protoplast is being grown, improved media and pre-culturing of the protoplast is recommended before placing this into micro-culture.
The protoplasts/cells have also been known to be cultured in semisolid media using different gelling agents such as agar, agarose, K-carrageenan, alginate, gelatin and polyacrylamide for secondary metabolite production. In this technique, the protoplast suspension at double the required density is gently mixed with double strength molten agar at 40-45xc2x0 C. in a petri dish so that protoplasts get embedded in the gel matrix upon solidification. However, a careful monitoring of agar gel temperature needs to be done to avoid damage to the protoplasts because of high temperature. The impurities present in agar also sometimes adversely affect the growth of the protoplasts.
Another method has been the use of semi-solid media using agarose (Lorz H., Larkin P. I., Thomson I. and Scowcroft W. R. 1983. Improved protoplast culture and agarose media. Plant Cell Tissue Org. Cult. 2: 217), wherein the protoplast suspension is made in agarose till micro-calli formation, which is then recovered by remelting the medium at 40xc2x0 C. But, reheating adversely affects cell growth. An improvement over this method is the use of alginate as the gelling agent (Adaoha-Mbanaso E. N. and Roscoe D. H. 1981. Alginate: an alternative to agar to plant protoplast culture. Plant Sci. Lett. 25: 61), which can then be dissolved in osmotically adjusted sodium citrate solution and the protoplast derived micro-colonies could be easily recovered without affecting the growth. However, another improvement of protoplast regeneration efficiency has been the use of a combination of both semi-solidified blocks/thin layers and liquid media. One such technique is bead culture method, wherein the protoplasts are suspended in agar or agarose. The gel blocks containing embedded protoplasts are transferred to liquid medium and placed on a shaker. In this manner, an improvement in the protoplast growth could be accomplished but again, this method is quite cumbersome and involves many steps. Out of all gelling agents, use of agarose has improved protoplast culture efficiency and also the production of secondary metabolites, when protoplasts are embedded in thin layers of agarose placed on top of the solidified medium in a petri dish. However, the cost and the handling of agarose is a major constraint. Moreover, the recovery of micro-colonies is difficult in the embedding procedure using agar or agarose as it requires heating and remelting of agar/agarose which may adversely affect the growth of protoplasts.
An improvement over all the methods described above is the the Thin Alginate Layer (TAL) technique (Golds T. J., Babezinsky J., Rauscher G. and Koop H. U. 1992. Computer controlled tracking of single cell development in Nicotiana tabaccum L. and Hordeum vulgare L. Protoplasts embedded in agarose/alginate films. J. Plant Physiol. 140: 582-587). This technique encompasses several positive points over earlier methods and was used for Nicotiana mesophyll protoplasts. In this method, squares of polypropylene mesh (2.0xc3x972.0 mm grid) are cut so that it exactly fits into 60 mm petridish. Protoplasts at twice the required density in MMM550 are suspended in equal volumes of 2.8% (w/v) alginic acid to give final alginate concentration of 1.4%. For one grid preparation, 625 xcexcl of this mixture is placed on an agar layer (20 mM CaCl2), and a polypropylene mesh (10xc3x9710 meshes; 2.0xc3x972.0 mm mesh size) is placed over it. After one hour, the grid is slid gently towards one side and taken out. This is then placed upside down in a 60 mm petri dish containing 10 ml of protoplast culture medium. Excess of calcium chloride is removed by giving two washings with protoplast culture medium (10 ml) and finally cultured in 2.0 ml of the same medium. These are then incubated in dark/diffused light at 25xc2x0 C. The protoplasts divide to form colonies. When colonies of 10-20 celled stage are formed, these are transferred to regeneration medium. Despite the fact that TAL technique has certain advantages like cell tracking, convenience in transfer to fresh medium, reduced release of toxic substances to the medium, it has certain limitations like
i) This process is time consuming and costly
ii) When grid is transferred to regeneration medium, callus or differentiated tissue sometimes folds upwards and ultimately slips out of grid, thereby loosing contact with the medium.
iii) The alginate layer is thick, hence protoplasts in multilayers are difficult to track.
iv) The use of a specific polypropylene mesh which is not easily available.
As seen above, all the methods described suffer from one or the other limitations, therefore, an improvement over TAL technique is the presently proposed Extra Thin Alginate Film (ETAF) technique.
Extra Thin Alginate Film Technique as compared to TAL technique is simpler to perform, less time consuming and economical. The advantages of this technique can be enumerated as follows:
a. It is less expensive than TAL technique as
agar component of CaCl2 is eliminated (25 ml of gelled CaCl2 medium is poured into petriplates where two grids can be prepared. For 25 ml of medium 200 mg of agar is used. Therefore for one grid 100 mg of agar is required).
Only 150 xcexcl of CaCl2 solution is required instead of 25 ml of CaCl2 medium. Similarly, for one coverglass only 50 xcexcl of Alginate is required.
Microslides and coverglass are less expensive, can be reused and are easily available throughout the world than the polypropylene grid.
Protoplast culture medium required for washing is just one ml for each washing compared to 10 ml in TAL technique.
This technique also saves time as compared to TAL technique as complexation time is only 5 minutes compared to one hour,
Two washings of 15 minutes each are required instead of two washings for 30 minutes,
preparation of grid (sizing, autoclaving and drying) consumes a lot of time, whereas microslide and coverglass can be sterilized after giving a dip in alcohol and by subsequent flaming,
e. Protoplasts are evenly spread and easy to track due to ultra thin layer,
f. While using TAL technique, callus or differentiated tissue sometimes folds upwards and ultimately slips out of the grid thereby loosing contact with the medium. However, in the present proposed ETAF technique none of these limitations prevail.
g. While performing fusion experiments on coverglasss, the protoplasts can be easily embedded using this technique.
Thus the present invention overcomes the limitations of all the protoplast culture techniques described so far.
The main objective of the present invention is to provide an efficient new method of protoplast culture.
Another objective of the present invention is to provide an extra thin embedding layer so that protoplasts are evenly spread for ease in observation and tracking for the purpose of plant genetic manipulations, particularly somatic cell hybridization, cybridization and transgenics.
Still another objective of the present invention is to simplify the procedure by using easily available merchandise such as glass microslides and coverglass and also to increase its universal appeal.
Yet another objective of the present invention is to reduce the cost factor involved and the time taken in protoplast culture.
Accordingly the present invention provides an efficient method for protoplast culture, which comprises the steps of:
a) isolating the protoplasts from a cell suspension,
b) mixing the protoplasts with equal volumes of alginate solution,
c) placing 40-50 xcexcl of CaCl2 solution on a clean glass microslide,
d) placing a mixture of protoplasts and alginate solution on the glass microslide and immediately covering by a glass coverglass,
e) adding CaCl2 solution in an amount of 70 to 100 xcexcl from the sides of coverglass,
f) sliding down of coverglass towards one side after four to ten minutes, with the help of jewellers forceps and placing in a petridish (Cell Culture Dish, 35xc3x9710 mm w/2 mm grid; Genetix, USA, Cat. No. 174926) containing one ml of protoplast culture medium,
g) sealing of petridishes with parafilm and incubating in dark/diffused light at 20 to 27xc2x0 C., and
h) transferring of Extra Thin Alginate Layer with 20-25 celled colonies to regeneration medium.
In an embodiment, rose (Rosa damascena) protoplasts from cell suspension were used.
In another embodiment, mesophyll protoplasts of Nicotiana tobacum cv. Petit Havana were used.
In yet another embodiment, cotyledon protoplasts of Lotus corniculatus were used.
In still another embodiment, the regeneration medium is modified MS medium supplemented with 0.1 mg/l Benzyl amino-purine and Naphthalene acetic acid 0.01 mg/l in nicotine.
In yet another embodiment, 40-50 xcexcl of CaCl2 solution is placed on a clean glass microslide.
In still another embodiment, mixture of protoplasts and alginate is placed on drop of CaCl2 and is immediately covered by a clean coverglass.
In yet another embodiment, more of CaCl2 solution is added from the sides of coverglass.
In yet another embodiment, the coverglass is gently slided down towards one side with the help of jewellers forceps.
The technique described in the invention can be performed by less expensive easily available material, wherein the callus or differentiated tissue remains constantly in touch with the nutritive medium and due to the extra thin layer, protoplasts are seen in one focus and are easy to track.
The advantages of the method of the invention over the prior art methods are provided hereinbelow and illustrated in FIG. 1 of the accompanying drawings.
The technique described can be performed by less expensive, easily available material, wherein the callus or differentiated tissue remains constantly in touch with the nutrition medium and due to extra thin layer protoplasts are seen in one focus and are easy to look.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention.